ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.

A liver-targeting Cu(i) chelator relocates Cu in hepatocytes and promotes Cu excretion in a murine model of Wilson's disease.

Copper chelation is the most commonly used therapeutic strategy nowadays to treat Wilson's disease, a genetic disorder primarily inducing a pathological accumulation of Cu in the liver. The mechanism of action of Chel2, a liver-targeting Cu(i) chelator known to promote intracellular Cu chelation, was studied in hepatic cells that reconstitute polarized epithelia with functional bile canaliculi, reminiscent of the excretion pathway in the liver. The interplay between Chel2 and Cu localization in these cells was demonstrated through confocal microscopy using a fluorescent derivative and nano X-ray fluorescence. The Cu(i) bound chelator was found in vesicles potentially excreted in the canaliculi. Moreover, injection of Chel2 either intravenously or subcutaneously to a murine model of Wilson's disease increased excretion of Cu in the faeces, confirming in vivo biliary excretion. Therefore, Chel2 turns out to be a possible means to collect and excrete hepatic Cu in the faeces, hence restoring the physiological pathway.

Membrane-enclosed multienzyme (MEME) synthesis of 2,7-anhydro-sialic acid derivatives.

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives.

The mucin-degradation strategy of Ruminococcus gnavus: The importance of intramolecular trans-sialidases.

We previously identified and characterized an intramolecular trans-sialidase (IT-sialidase) in the gut symbiont Ruminococcus gnavus ATCC 29149, which is associated to the ability of the strain to grow on mucins. In this work we have obtained and analyzed the draft genome sequence of another R. gnavus mucin-degrader, ATCC 35913, isolated from a healthy individual. Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans. R. gnavus ATCC 35913 also encodes a predicted IT-sialidase and harbors a Nan cluster dedicated to sialic acid utilization. We showed that the Nan cluster was upregulated when the strains were grown in presence of mucin. In addition we demonstrated that both R. gnavus strains were able to grow on 2,7-anyhydro-Neu5Ac, the IT-sialidase transglycosylation product, as a sole carbon source. Taken together these data further support the hypothesis that IT-sialidase expressing gut microbes, provide commensal bacteria such as R. gnavus with a nutritional competitive advantage, by accessing and transforming a source of nutrient to their own benefit.